The broad long-term objectives of the proposed research are to characterize the interaction of HIV nef with its cellular binding partners and to understand the functional consequences of this interaction for HIV infection and immunity. The proposed research focuses on the interaction of nef with several T cell-surface proteins: the T cell antigen receptor (TCR), MHC class I molecules and the related CD1 antigen, the CD4 co-receptor, and the CD28 co- stimulatory molecule. These proteins comprise the currently known complement of cell- surface targets for HIV nef. Each of these proteins is involved in the activation of T cell immune responses, and each is known to be down-modulated by interaction with nef. For the T cell antigen receptor, interaction with nef is known also to lead to modulation of T cell signaling pathways. Currently, little is known about the structural basis for the interaction of nef with these targets, and a major goal of the proposed research is structural characterization of these interactions. For each of these receptors, the presumptive nef binding partner is a believed to be an unstructured or partially-folded cytoplasmic domain, and an important component of the proposed research is the development of methods for the analysis of interactions among proteins with natively unfolded or loosely-folded domains. The proposed research includes two specific aims and the development of a set of shared analysis tools. The first aim is to characterize the interaction of HIV nef with the TCR zeta subunit cytoplasmic domain, including measurements of the affinity, kinetics, and stoichiometry of the interaction, the HIV strain and subtype dependence, determination of the three-dimensional structure of the complex, investigation of binding-induced structural changes in both nef and the TCR zeta domain, characterization of the mechanism of TCR signaling modulation by nef. The second aim is to extend these studies to other cell surface receptor binding targets including MHC, CD1, CD4, and CD28. A common methodology will be developed to overcome challenges for structural and functional characterization in this and other systems involving unstructured for partially-structured protein domains. The proposed research is structured in two phases. The R21 phase (years 1 and 2) includes the refinement of tools for structural characterization of nef-target interactions and for evaluation of the functional outcome of these interactions, the application of these methods to the interaction of HIV-2/SIV nef with the TCR zeta subunit cytoplasmic domain, and characterization of basic binding parameters for the interaction of HIV nef with CD4, MHC class I, CD1a, CD1d, and CD28. The R33 phase (years 3-5) includes the application of the structural and functional tools to the targets in the latter set for which clear evidence of specific nef interaction is obtained, and the detailed evaluation of the strain/subtype dependence of these interactions. Nef is important for the virulence of HIV, but the molecular details of how nef interacts with its binding partners in an infected cell are not clear. In the proposed research, these interactions will be studied using biochemical and biophysical approaches. This information will promote efforts to understand how nef modifies the signaling machinery in an infected cell, and to develop therapeutic approaches to interfere with this aspect of HIV infection and pathogenesis. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]